Streptavidin, which is an avidin homolog from the bacterium Streptomyces avidinii, is used broadly in science and engineering because it can bind biotinylated ligands with high affinity. The interaction is robust, context independent and simple to implement. For example, many commonly used protein reagents (e.g. ligands, enzymes and antibodies) are already available in biotinylated forms and can be used readily in (strept)avidin-based detection systems. Streptavidin also has high thermodynamic and chemical stability and can be conjugated with organic molecules or enzymes without loss of function, which is an important attribute for its use in different experiments. Common uses of streptavidin include detection and quantification of biotinylated ligands, as well as immobilization, crosslinking, purification, and labeling of various biotinylated targets. Other uses of streptavidin include fluorescence microscopy or flow cytometric analysis of cells, in which cell surface proteins are enzymatically biotinylated or localized with biotinylated ligands, toxins or antibodies for subsequent labeling with fluorescent streptavidin.
However, labeling of biotinylated targets on live cells with wild type streptavidin (wtSA) can be problematic because of its tetrameric structure, which can result in aggregation of target molecules that interferes with measurements at a molecular level. Target aggregation can be avoided by using monovalent streptavidin, as was shown using an engineered monovalent tetramer containing a single active binding site. Although monovalent tetramer binds biotinylated receptors without target clustering, preparation of the molecule requires in vitro folding and lengthy biochemical separation steps. To simplify monovalent detection of biotinylated targets, an engineered monomeric streptavidin (mSA) has been generated (Lim et al., Biotechnology Bioeng. 110, 57-67, 2013). Despite its useful structural and biochemical properties, currently available mSA, including the one described in Lim et al., have rapid biotin dissociation kinetics (koff=1.05×10−3 s−1 or dissociation t1/2=11 min). As such, cells labeled with fluorophore conjugated mSA lose fluorescence over time, resulting in time dependent signal loss that complicates detection and analysis. In this regard, engineering an mSA mutant with a slower dissociation rate is needed to increase its performance in some biotechnology applications, including live cell imaging.
Beyond the above, one of the challenges when using mSA in experiments is the difficulty of its purification. Existing mSA purification protocols are based on inclusion body purification or use a solubilization tag, e.g. maltose binding protein or thioredoxin. Both methods are slow, expensive and require in vitro folding. The difficulty of preparing mSA limits usefulness of the molecule—with or without improvement in the binding characteristics—especially if the end user is unable to prepare the material for a planned study.
There remains a need for a stable, high affinity monomeric streptavidin possessing a slow dissociation rate and which can be easily prepared.